Nitazoxanide

International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 98–105

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Nitazoxanide: In vitro and in vivo drug effects against Trichuris muris and Ancylostoma ceylanicum, alone or in combination ⇑ Lucienne Tritten 1, Angelika Silbereisen 1, Jennifer Keiser

Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, University of Basel, Basel, Switzerland article info abstract

Article history: Soil-transmitted helminths cause more than 1 billion human infections globally, mostly in the poorest Received 28 January 2012 regions of the world. Control relies essentially on a limited panel of four drugs, and drug resistance might Received in revised form 24 February 2012 be inescapable. Nitazoxanide, an anti-infective drug, has been shown to exert anthelmintic activity in Accepted 26 February 2012 human clinical trials. In the present work, nitazoxanide was tested alone or combined with commercial- Available online 10 March 2012 ized anthelmintics on Trichuris muris, a whipworm mouse model, and Ancylostoma ceylanicum, a hook-

worm hamster model, in vitro and in vivo.IC50sof61 and 12.87 lg/ml were achieved with Keywords: nitazoxanide on T. muris third-stage larvae (L3) and adult worms in vitro, respectively. An IC of Trichuris 50 61 g/ml was obtained exposing A. ceylanicum adults worms to nitazoxanide, whereas A. ceylanicum Ancylostoma l Nitazoxanide L3 were not affected. Using scanning electron microscopy, the tegument of adult T. muris appeared Drug combination unchanged following nitazoxanide treatment, whereas swellings were seen on the tegument of the anterior region of half of the A. ceylanicum specimen analyzed. Synergism was observed in vitro when nitazoxanide was combined with levamisole or ivermectin on T. muris adult worms, and when combined with levamisole, pyrantel pamoate, or ivermectin on A. ceylanicum adult worms. In T. muris-infected mice, oral nitazoxanide achieved worm burden reductions of 56.09% and 17.37% following a single dose of 100 mg/ kg and three doses of 50 mg/kg, respectively. None of the tested drug combinations displayed activity on T. muris in vivo.InA. ceylanicum-infected hamsters, no effect was observed for oral nitazoxanide alone, and none of the tested combinations reached the threshold for additive effect. In conclusion, nitazoxanide failed to demonstrate promising activity against T. muris and A. ceylanicum in vivo, regardless whether tested as monotherapy or combined with standard drugs. Reasons for the discrepancy of these ﬁndings compared to results obtained in clinical trials remain to be elucidated. Ó 2012 Australian Society for Parasitology Published by Elsevier Ltd. All rights reserved.

1. Introduction (Bethony et al., 2006; Mascarini-Serra, 2011). The arsenal of available drugs for the treatment of STH infections is limited: Soil-transmitted helminths (STH) are parasitic intestinal nema- albendazole, mebendazole, levamisole, and pyrantel pamoate are todes affecting more than one billion people worldwide (de Silva recommended by the World Health Organization (WHO, 2011). et al., 2003; Bethony et al., 2006; Hotez et al., 2008). The diseases Almost all human anthelmintics were originally developed for vet- caused by STHs belong to the so-called neglected tropical diseases, erinary needs and have not been optimized for human use (Geary because they receive only a small percentage (<1%) of global re- et al. 2010). This is reﬂected in low cure rates, in particular when search funding (de Silva et al., 2003; Bethony et al., 2006; Hotez single oral doses are administered against Trichuris trichiura infec- et al., 2007). Pre-school and school-aged children, pregnant women tions (Keiser and Utzinger, 2008, 2010). and fetuses are most vulnerable and may suffer profound morbid- In addition, anthelmintic resistance is widespread among nem- ities, including anemia, growth impairment and mental retardation atodes of livestock as a consequence of frequent drug administra- (Bethony et al., 2006; Hotez et al., 2007). The current global ap- tion of the same class of compounds over long periods (Geerts proach to control helminth infections is based on chemotherapy, et al., 1997; Wolstenholme et al., 2004). Though there is no conclu- mainly administered in the frame of school deworming campaigns sive evidence of anthelmintic resistance in humans, there is an ur- gent need to develop backup drugs. However, despite the large global burden of human STH (Bethony et al., 2006), few efforts have been made to discover and develop novel nematocidal drug ⇑ Corresponding author. Address: Department of Medical Parasitology and candidates (Keiser and Utzinger, 2010; Olliaro et al., 2011). In addi- Infection Biology, Swiss Tropical and Public Health Institute, P.O. Box, CH-4002 tion to the discovery and development of novel antiparasitic drugs, Basel, Switzerland. Tel.: +41 61 284 8218; fax: +41 61 284 8105. E-mail address: [email protected] (J. Keiser). combination chemotherapy is a powerful strategy to slow emer- 1 The authors contributed equally to this work. gence of drug resistance (Barnes et al., 1995; Nyunt and Plowe,

2211-3207/$ - see front matter Ó 2012 Australian Society for Parasitology Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpddr.2012.02.004 L. Tritten et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 98–105 99

2007). Therefore, combinations with both new and marketed drugs dose of 200 embryonated T. muris eggs (Wakelin, 1970; Stepek have to be explored. et al., 2006). Hamsters were infected orally with 150 A. ceylanicum Nitazoxanide, first described in the 1980s, is a broad-spectrum third-stage larvae (L3) (Ray and Bhopale, 1972; Garside and thiazolide compound with anthelmintic, antiprotozoal and antivi- Behnke, 1989). Hamsters assigned to in vivo studies were not ral properties (Rossignol and Maisonneuve, 1984; Rossignol and immunosuppressed and were infected with 300 L3. Stachulski, 1999; Hemphill et al., 2006). More recently, it has been demonstrated that nitazoxanide exhibits also activity when 2.4. In vitro studies administered in multiple doses against a number of geohelminths, including Ascaris lumbricoides and T. trichiura (Fox and Saravolatz, 2.4.1. T. muris 2005; Anderson and Curran, 2007; van den Enden, 2009). Nitazox- 2.4.1.1. Activity of nitazoxanide on L3 and adults. In vitro studies anide was therefore listed as a potential drug development candi- with T. muris were performed as described previously (Stepek date eligible for rapid transitioning into development for human et al., 2006; Silbereisen et al., 2011). Briefly, L3 (on day 20 p.i.) STH infections (Olliaro et al., 2011). and adult worms (from day 35 p.i. onwards) were isolated from Hence, it was our aim to further characterize the potential of the intestines of mice (Stepek et al., 2006). Three to four parasites nitazoxanide as nematocidal drug using the parasite-rodent mod- were incubated for 72 h at 37 °C, 5% CO2 in 100 ll (96-well) or els Trichuris muris and the hookworm Ancylostoma ceylanicum. In 500 ll (48-well) RPMI medium [10.44 g RPMI 1640 (Gibco, Basel, vitro, the worms’ viability of larvae and adult worms was deter- Switzerland), 5 g albumax H (Gibco), 5.94 g HEPES (Sigma–Al- mined following incubation with nitazoxanide using the motility drich), 2.1 g sodium bicarbonate (Sigma–Aldrich), in 1 l dH2O sup- assay (Stepek et al., 2006; Kopp et al., 2008; Silbereisen et al., plemented with 5% v/v amphotericin B (250 lg/ml, Sigma–Aldrich) 2011). Scanning-electron microscopy pictures are presented to and 1% v/v penicillin–streptomycin (10,000 U/ml penicil- underscore our results. In vivo, T. muris-infected mice and A. ceylan- lin + 10 mg/ml streptomycin, Sigma–Aldrich), in the presence of icum-infected hamsters were treated with single or multiple doses 12.5, 25, 50, 100 and 200 lg/ml (adults) and 50, 100 and 200 lg/ of nitazoxanide. In addition, the drug was combined in vitro with ml (L3) nitazoxanide (Fonseca-Salamanca et al., 2003). Parasite each standard drug (albendazole, mebendazole, levamisole, pyran- motility was evaluated microscopically (Carl Zeiss, Germany, mag- tel pamoate) as well as ivermectin and the interaction of each com- nification 20–80Â) 24, 48 and 72 h after start of incubation, using a bination was assessed. Synergistic combinations were tested motility scale from 3 (normal motility, full viability) to 0 (no move- in vivo. ment, death). As controls, 3 worms incubated in the highest DMSO concentration used in the assays and in RPMI medium alone were used (2% v/v). 2. Materials and methods

2.4.1.2. Combination chemotherapy studies. Nitazoxanide was 2.1. Drugs combined with albendazole, mebendazole, levamisole, pyrantel pamoate or ivermectin using a constant dose ratio (DR) based Nitazoxanide was kindly provided from Laboratoria Wolfs on estimated IC values: nitazoxanide = 25 lg/ml, albendazole = (Zwijndrecht, Belgium) and for in vitro assays a drug stock (5– 50 200 lg/ml, mebendazole and ivermectin = 100 lg/ml, levamisole 10 mg/ml) was prepared in 100% DMSO (Sigma–Aldrich, Buchs, and pyrantel pamoate = 50 lg/ml (Tritten et al., 2011 and unpub- Switzerland) and stored at 4 °C. For in vivo assays, a nitazoxanide lished work). Three to four adult T. muris were incubated in four suspension in 10% [Tween 80, 80% EtOH (70:30 v/v, Fluka)] and different concentrations of each drug combination (2xIC : 2xIC , 90% dH O was prepared before treatment. Albendazole, mebenda- 50 50 2 IC :IC , 0.5xIC : 0.5xIC and 0.25xIC : 0.25xIC ). zole, pyrantel pamoate and ivermectin were purchased from Sig- 50 50 50 50 50 50 ma–Aldrich, and levamisole hydrochloride, from Fluka (Buchs, 2.4.2. A. ceylanicum Switzerland), and prepared in the same way. 2.4.2.1. Activity of nitazoxanide on L3 and adults. In vitro studies with A. ceylanicum were conducted as described recently (Tritten 2.2. Animals and parasites et al., 2011, 2012). Briefly, 30 L3 per well (96-well plates, Costar) were incubated for 72 h at room-temperature in 200 ll HBSS med- Three to five week-old female C57BL/10 mice and 3 week-old ium (Gibco) supplemented with 10% v/v amphotericin B (250 lg/ male Syrian golden hamsters were purchased from Charles River ml, Sigma–Aldrich) and 1% v/v penicillin–streptomycin (10,000 U/ (Blackthorn, UK and Sulzfeld, Germany, respectively). Before infec- ml penicillin + 10 mg/ml streptomycin, Sigma–Aldrich) containing tion, animals were allowed to acclimatize for 1 week, kept in drug concentrations ranging from 0.01 to 100 lg/ml. The larval groups of 10 (mice) or 5 (hamsters) in macrolon cages with free ac- survival (ability to move, alive; no movement, death) was investi- cess to water and rodent food pellets (Rodent Blox from Eberle NA- gated microscopically (magnification 20Â) following addition of FAG, Gossau, Switzerland) and with a 12 h light/dark cycle hot water (80 °C) and exposure to microscope light. Drug suscep- according to Swiss cantonal and national regulations (Permission tibilities of adult worms (obtained by dissection of hamster intes- No. 2070). T. muris was obtained from Prof. J.M. Behnke (University tines) were tested in 48-well plates (Costar) with 2–3 worms per of Nottingham) and Prof. H. Mehlhorn (University of Düsseldorf) in well and 1 ml HBSS medium supplemented with antibiotics and 2010 and A. ceylanicum was provided by Prof. J.M. Behnke (Univer- 10% v/v fetal calf serum (Connectorate AG, Switzerland) in the sity of Nottingham), in 2009. presence of 0.01–100 lg/ml of the drugs at 37 °C, 5% CO2 for 72 h. The motility was determined microscopically (magnification 2.3. Parasites and infections 20Â) using a viability scale ranging from 2 (normal motility, full viability) to 0 (no movement, death). Based on the motility values

The T. muris and A. ceylanicum rodent models have been de- obtained with L3 and adults IC50s were calculated. Control worms scribed elsewhere (Tritten et al., 2011). Mice and hamsters were were incubated in the highest DMSO concentration used in the test immunosuppressed with dexamethasone in the drinking water (2% v/v). Assays were conducted in duplicate at least twice. (4 mg/l and 1 mg/ml, respectively, dexamethasone-water soluble, Sigma–Aldrich) from 2 days before infection until 2 days before 2.4.2.2. Combination chemotherapy studies. L3 and adult worms treatment (Campbell, 1968; Wakelin, 1970). Mice received an oral were incubated with nitazoxanide combined with albendazole, 100 L. Tritten et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 98–105 mebendazole, levamisole, pyrantel pamoate or ivermectin, as de- Briefly, the fecal egg burden was established on days 21 and scribed recently (Tritten et al., 2012). Briefly, 50 ll of the individual 22 p.i. and hamsters were assigned to equally balanced treatment drug solutions were added to each well and serially diluted, in or- groups accordingly (3–4 animals each). Hamsters were treated der to have final concentrations ranging from 2ÂIC50 to 0.25ÂIC50 with a single oral dose of 10 mg/kg nitazoxanide on day 23 p.i. Four for each drug. IC50 values of the partner drugs have been presented animals were left untreated and served as controls. Stools were recently (Tritten et al., 2011, 2012). The IC50 values of mebendazole collected from each hamster over a period of 48 h after treatment were determined in the present work. The assay was conducted at and carefully searched for worms. Hamsters were sacrificed on day least twice in duplicate and the interaction of each combination 7 post-treatment and the remaining worms counted. WBR and was assessed. WER were calculated (Rajasekariah et al., 1986; Xue et al., 2005; Tritten et al., 2011). 2.4.2.3. Ovicidal activity of nitazoxanide. The ovicidal activity of nitazoxanide was assessed following a modified protocol based 2.5.2.2. Combination chemotherapy studies. Combinations behaving on Fonseca-Salamanca et al. (2003) and Tritten et al. (2011). synergistically at IC50 in vitro were tested in vivo. Nitazoxanide Briefly, in 48-well plates (Costar), 50 eggs were incubated in (10 mg/kg) was combined with levamisole, pyrantel pamoate or dH2O containing 10 lg/ml nitazoxanide. After 24 h exposure at ivermectin. Approximate ED50 values were calculated for the part- room-temperature, 20 eggs per well were examined microscopi- ner drugs (levamisole: 10 mg/kg (Tritten et al., 2012), pyrantel cally (magnification 80–160Â) for embryonation, and after 48 h, pamoate: 10 mg/kg, ivermectin: 0.04 mg/kg (unpublished data)). for hatching. Control wells contained the same amount of DMSO used as in the drug assay (0.2% v/v). The assay was conducted in 2.6. Statistical analyses quadruplicate.

The average of motility scores was calculated for each concen- 2.4.3. Scanning electron microscopy studies tration, converted into a percentage value and normalized to the The effect of nitazoxanide on the tegument of adult T. muris and control, using MicrosoftÒ Excel 2010. Differences in the motility A. ceylanicum was observed using scanning electron microscopy of drug-treated and control worms and ovicidal activity were (SEM). After 24 h of incubation with the drug, both parasites were examined using the Fisher’s exact test, with a significance level fixed in 2.5% glutaraldehyde (Alfa Aesar GmbH, Germany) in PBS of 0.05, using Microsoft Excel 2010 or StatsDirect (version 2.4.5; (pH 7.4) for approximately 24 h at room-temperature. Each worm StatsDirect Ltd., Cheshire, UK). IC values were expressed based was washed three times in PBS and stored in 500 ll PBS at 4 °C un- 50 on the median effect principle using the CompuSyn software (ver- til use. Before SEM examination, the samples were dehydrated sion 1.0), where a fitted curve is modeled and r is the linear corre- stepwise for 10 min in 500 ll ascending ethanol concentrations lation coefficient of the median-effect plot, indicating the goodness (30%, 50%, 70% and >96% (ethanol absolute, Merck)), at room-tem- of fit (Chou, 1976). Combination indices (CI) were calculated with perature and kept in 500 ll 96% ethanol at 4 °C(Manneck et al., CompuSyn. Briefly, the behavior of a combination was categorized 2010). Finally, the worms were dried to critical point (Bal-tec according to the following scale: CI > 1: antagonism; CI = 1: addi- CPD 030), fixed on aluminum stubs and sputter coated with tive effect; CI < 1: synergism (Chou, 1976). Worm burden reduc- 20 nm gold particles. SEM pictures were acquired with a high-res- tions were determined by comparing the mean number of adult olution scanning electron microscope (Phillips XL30 ESEM and worms in the intestine of a treated group with the mean number Nova™ Nano SEM 230) at an accelerating voltage of 5 kV. of the control group, in MicrosoftÒ Excel 2010. Worm expulsion rates were calculated by dividing worm output of a treatment 2.5. In vivo studies group by the total worm burden, using MicrosoftÒ Excel 2010. ED s were calculated based on the WBRs, using CompuSyn. The 2.5.1. T. muris 50 Kruskal–Wallis test (multiple doses against control) or the 2.5.1.1. Nitazoxanide monotherapy. In vivo studies with T. muris-in- Mann–Whitney U test (single dose against control) was used to as- fected mice were conducted as described recently (Tritten et al., sess the statistical significance of the WBRs, using StatsDirect. 2011). Briefly, each mouse was checked for presence of infection (presence of eggs in the stools) on day 41 p.i. Groups of 3–4 infected animals were assigned to treatment (100, 300, 600 mg/kg 3. Results single dose or 50 mg/kg nitazoxanide per day over 3 days (Fonse- ca-Salamanca et al., 2003) or control groups (4 animals each). Ex- 3.1. In vitro studies pelled worms present in the mouse stools (collected up to 72 h after treatment) were counted. On day 7 post-treatment, the mice 3.1.1. T. muris were sacrificed and the remaining worms in the gut counted. The 3.1.1.1. Activity of nitazoxanide on L3 and adults. Nitazoxanide had a worm burden reduction (WBR) as well as the worm expulsion rate marked effect on both T. muris L3 and adult worms. L3 were either (WER) were calculated for each treatment (Rajasekariah et al., dead (200, 50 lg/ml) or showed a large reduction in viability 1991). (reduction >85%, all P < 0.001) following incubation with 100 lg/ ml for 72 h (data not shown). The temporal effect of five different 2.5.1.2. Combination chemotherapy studies. Combinations found concentrations of nitazoxanide on the viability of adult T. muris is synergistic at IC50 in vitro were tested in vivo. Drugs were com- presented in Fig. 1. Adults incubated in nitazoxanide (200, 100, bined using a constant ratio based on the approximate ED50 values 50 lg/ml) were markedly affected (>65% viability reduction) 24 h of the partner drugs (unpublished data). Nitazoxanide (100 mg/kg, post-incubation (all P < 0.001). At 200 lg/ml adult T. muris were best-performing dose) was combined with levamisole (46 mg/kg) dead after 48 h (P < 0.001). After 72 h, adult T. muris were still alive or ivermectin (4 mg/kg). Drug effects were analyzed as described when exposed to 100 and 50 lg/ml nitazoxanide, but showed little above. movement (motility <1, all P < 0.001). Nitazoxanide concentrations of 25 lg/ml reduced viability by 44% (P < 0.03), whereas a concen- 2.5.2. A. ceylanicum tration of 12.5 lg/ml of the drug had no effect on adult worms 2.5.2.1. Nitazoxanide monotherapy. The A. ceylanicum experiments (P > 0.05) 72 h post-incubation. All control worms showed normal were carried out as summarized recently (Tritten et al., 2011). movement (motility = 3) during the entire incubation period L. Tritten et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 98–105 101

(motility values of 1.6 and 1.75, respectively (both P > 0.05)). An

IC50 of 0.74 lg/ml (r = 0.93) was determined (Table 1). For comparison, control worms incubated with DMSO showed an average motility of 1.93. No reduction in egg embryonation and hatching was observed in nitazoxanide-treated wells, compared to the controls (both P = 0.50).

3.1.2.2. Combination chemotherapy studies. Nitazoxanide combined with albendazole resulted in a CI of 1.90, suggesting an antagonistic behavior against adult worms in vitro (Table 1). An additive effect (CI = 1.0) was observed with the nitazoxanide–mebendazole combination. Nitazoxanide combined with levamisole, pyrantel pamoate or ivermectin showed synergism, with CIs of 0.32, 0.14 and 0.69, respectively. Fig. 1. In vitro effect of nitazoxanide on adult T. muris worms after 24, 48 and 72 h of incubation with 12.5, 25, 50, 100 and 200 lg/ml. NTZ = nitazoxanide. Motility 3.1.3. Scanning electron microscopy studies scale: 3 = normal motility; 2 = low motility; 1 = very low motility; 0 = dead. 3.1.3.1. T. muris. Nitazoxanide treated T. muris (200 lg/ml) and control worms were examined after incubation for 24 h (Fig. 3A–

(72 h). IC50s of 0.27 lg/ml (r = 0.92) and 12.87 lg/ml (r = 0.70) D). According to our in vitro studies worms are already strongly af- were calculated for L3 and adults, respectively. IC50s are summa- fected at this examination point. rized in Table 1. No tegumental changes were observed with treated worms (Fig. 3A and C) compared to controls (Fig. 3B and D) at the anterior as well as the posterior tegument. 3.1.1.2. Combination chemotherapy studies. Five different drug combinations were tested against adult T. muris. A synergistic effect was 3.1.3.2. A. ceylanicum. In 2 out of 4 specimen, SEM revealed many observed with the combination of nitazoxanide and ivermectin areas of swelling (knobs) in the anterior part of adult A. ceylanicum (CI = 0.63, DR = 1:4). A nearly additive effect was calculated for nita- 24 h post-incubation in the presence of a lethal concentration of zoxanide combined with levamisole (CI = 0.99, DR = 1:2). For exam- 100 lg/ml nitazoxanide (all worms were dead, Fig. 4A). These ple, even the lowest concentrations of nitazoxanide (6.25 lg/ml) structures were not observed on the tegument of control worms and levamisole (25 lg/ml) tested reduced the viability of the worms that had been incubated with DMSO (Fig. 4B). to less than 50% (motility: 1.29 ± 0.49, P < 0.001). The combinations of nitazoxanide–pyrantel pamoate (DR = 1:2), nitazoxanide–alben- 3.2. In vivo studies dazole (DR = 1:8), and nitazoxanide–mebendazole (DR = 1:4) revealed antagonistic effects (CI = 4.94, 2.64 and 2.28, respectively). 3.2.1. T. muris Normal viability was observed with control worms (motility = 3) 3.2.1.1. Nitazoxanide monotherapy. Mice were treated with single until the end of the experiment (72 h). 100, 300 or 600 mg/kg nitazoxanide, or 3 doses of 50 mg/kg given over 3 consecutive days (Table 2). The highest WBR of 56.09% was 3.1.2. A. ceylanicum achieved following administration of 100 mg/kg nitazoxanide. 3.1.2.1. Activity of nitazoxanide on L3, adults and eggs. No effect on However, a low WER of 16.23% was observed for this dose. Higher L3 motility was observed when A. ceylanicum were incubated with dosages did not result in increased activity (WERs of 2.0% and 0% 100 lg/ml nitazoxanide (P = 0.50) for 72 h. As many as 74.2% of lar- and WBRs of 0% and 0%, at 300 and 600 mg/kg, respectively). Worm vae survived in the presence of 10 lg/ml (P < 0.001), 81.1% in 1 lg/ burden reductions in nitazoxanide-treated mice (all single dos- ml (P < 0.001) and 93.3% in 0.1 lg/ml (P = 0.059) (Fig. 2). An IC50 of ages) were not signiﬁcant (P = 0.910). Fifty mg/kg given in multiple >100 lg/ml (r not determined) was calculated for L3 (Table 1). doses reduced the worm burden by 17.37% (P = 0.543) and resulted Adult worms were very sensitive to nitazoxanide. A concentra- in a WER of 1.84%. tion of 100 lg/ml killed all adult worms (P < 0.001). At 10 lg/ml, the worms showed strongly reduced viability (motility of 0.8; 3.2.1.2. Combination chemotherapy studies. Nitazoxanide (100 mg/

P = 0.028). The lower nitazoxanide concentrations of 1 and kg) combined with levamisole (ED50) resulted in a moderate WER 0.1 lg/ml only slightly decreased adult hookworm motility of 33.9% and a very low WBR (0.27%) (P = 0.629). Nitazoxanide

Table 1

Median effect doses (IC50s) of nitazoxanide, mebendazole and ivermectin and combination indices of selected drug combinations on T. muris and A. ceylanicum.

Drug IC50s(r)(lg/ml) T. muris A. ceylanicum L3 Adults L3 Adults Nitazoxanide 0.27 (0.92) 12.87 (0.70) >100 (n.d.) 0.74 (0.93) Mebendazole n.d. n.d. 11.55 (0.87) >100 (0.87)

CI at IC50 nitazoxanide–albendazole n.d. 2.64 n.d. 1.90

CI at IC50 nitazoxanide–mebendazole n.d. 2.28 n.d. 1.00

CI at IC50 nitazoxanide–levamisole n.d. 0.99 n.d. 0.32

CI at IC50 nitazoxanide–pyrantel pamoate n.d. 4.94 n.d. 0.14

CI at IC50 nitazoxanide–ivermectin n.d. 0.63 n.d. 0.69

IC50s were calculated 72 h post incubation; r = linear correlation coefficient of the median-effect plot, indicating the goodness of fit. r P 0.85 indicates a satisfactory fit. n.d.: not determined. 102 L. Tritten et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 98–105

Fig. 2. Comparison of the in vitro effect of nitazoxanide on different parasite stages of A. ceylanicum. (A) L3; (B) adults. NTZ = nitazoxanide. Black bars represent incubation with 100 lg/ml, white bars incubation with 10 lg/ml, striped bars incubation with 1 lg/ml and grey bars incubation with 0.1 lg/ml. Motility scale for adults: 2 = normal motility; 1 = low motility; 0 = dead.

Fig. 3. SEM pictures of T. muris adults incubated for 24 h with nitazoxanide (A + C) or medium without drug (B + D). (A) Tegument of a nitazoxanide-treated worm (200 lg/ ml) and the corresponding control (B), (both magniﬁcation 1000Â), (C) anterior part with glands of nitazoxanide-treated worms and control (D) both at higher magniﬁcations (3000Â).

combined with ivermectin at the same ratio achieved a WER of 25% 3.2.2.2. Combination chemotherapy studies. Levamisole (10 mg/kg) and had no effect on the WBR (P = 0.629). administered simultaneously with nitazoxanide (10 mg/kg) pro- duced a WER of 64.29% and a low WBR of 18.02% (P = 0.80) 3.2.2. A. ceylanicum (Table 3). The combination nitazoxanide–pyrantel pamoate (both 3.2.2.1. Nitazoxanide monotherapy. Treatment of A. ceylanicum-in- 10 mg/kg) moderately reduced the infection, with an observed fected hamsters with a single 10 mg/kg oral dose had no effect WER of 27.78% and a WBR of 70.27% (P = 0.60). Ivermectin on the worm burden (WER and WBR = 0%) (P = 0.857), as shown administered together with nitazoxanide (0.04 mg/kg + 10 mg/kg, in Table 3. respectively) resulted in a WER of 56.34% and a WBR of 70.19% L. Tritten et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 98–105 103

Fig. 4. SEM pictures of A. ceylanicum adult worms. (A) Treated with nitazoxanide, anterior part showing tegumental knobs (arrow) (magniﬁcation 1000Â), (B) anterior part of a control worm (magniﬁcation 1000Â).

Table 2 In vivo effect of nitazoxanide on T. muris, alone and in combination.

Group Dose (mg/kg) Mean number of worms (SD) Mean number of expelled worms (SD) Worm expulsion rate Worm burden reduction (%) P-value (%) Control 1 – 109.5 (32.91) 0.68 (0.50) 0 – – Control 2 – 157.5 (62.57) 0.2 (0.45) 0 – – Control 3 – 98.33 (28.04) 0.33 (0.58) 0.34 – – Control 4 – 56.0 (55.32) 0 (0) 0 – – Nitazoxanide 1001 57.0 (60.55) 9.25 (13.20) 16.23 56.09 0.910a 3003 182.5.0 (114.54) 3.66 (6.35) 2.0 0 6004 87.0 (74.48) 0 (0) 0 0 50 Â 32 122.0 (32.84) 2.25 (3.30) 1.84 17.37 0.543b Control 5 – 91.25 (23.73) 0 (0) 0 – – Nitazoxanide– 100 + 465 137.66 (99.80) 46.67 (37.16) 33.90 0.27 0.629b levamisole Nitazoxanide– 100 + 45 185.33 (150.51) 46.33 (21.96) 25.0 0 0.629b ivermectin

Numbers in superscript refer to the corresponding control group. a Kruskal–Wallis test comparing the median of the worm burdens of control and treated mice. b Mann–Whitney U test comparing the median of the worm burdens of control and treated mice.

Table 3 In vivo effect of nitazoxanide on A. ceylanicum, alone and in combination.

Group Dose (mg/ Mean number of worms Mean number of expelled worms Worm expulsion rate Worm burden P- kg) (SD) (SD) (%) reduction (%) valuea Control 1 – 13.8 (8.0) 0 (0) 0 – – Nitazoxanide 101 17.8 (2.10) 0 (0) 0 0 0.857 Control 2 – 18.5 (23.33) 0 (0) 0 – – Control 3 – 26.0 (4.24) 0 (0) 0 – – Nitazoxanide–levamisole 10 + 102 9.33 (8.10) 6.0 (4.60) 64.29 18.02 0.80 Nitazoxanide–pyrantel 10 + 102 18.0 (20.10) 5.0 (2.70) 27.78 70.27 0.60 pamoate Nitazoxanide–ivermectin 10 + 0.043 17.75 (15.80) 10 (5.89) 56.34 70.19 0.80

Numbers in superscript refer to the corresponding control group. a Mann–Whitney U test comparing the median of the worm burdens of control and treated hamsters.

(P = 0.80). Hence, none of the drug combinations tested achieved a Nitazoxanide was first described for its cestocidal properties, la- higher efficacy than levamisole, pyrantel pamoate or ivermectin ter for anti-protozoal and anti-viral activities and recently for its po- alone (Tritten et al., 2011). tential against different nematodes and trematodes (Rossignol and Maisonneuve, 1984; Anderson and Curran, 2007; van den Enden, 2009). Among the helminths, Fasciola hepatica (Favennec et al., 4. Discussion 2003), A. lumbricoides, T. trichiura, Ancylostoma duodenale, Enterobius vermicularis and Strongyloides stercoralis have been shown in clinical Worldwide, about 3 billion people are estimated to be at risk of trials to be affected by treatment with nitazoxanide (Romero Cabello STH infections (de Silva et al., 2003). Since we rely on only four drugs et al., 1997; Abaza et al., 1998; Ortiz et al., 2002). for the treatment of STH infections, many of which are suboptimal, In the present investigation, we evaluated the activity of nita- new therapies and/or drug combinations are urgently needed. zoxanide against T. muris and A. ceylanicum, two well-established 104 L. Tritten et al. / International Journal for Parasitology: Drugs and Drug Resistance 2 (2012) 98–105 parasite-rodent models that mimic human soil-transmitted hel- and Hemphill, 2011). It has been suggested that the activity of nita- minthiases, in vitro and in vivo. With minor differences in sensitiv- zoxanide is quenched in the stomach by gastric acids (pH 3), by ity, both T. muris stages were markedly affected by nitazoxanide transforming the drug into a biologically inactive protonated form in vitro. Adult worms incubated with 50, 100 and 200 lg/ml nita- which might be restored to the active anion by the more alkaline zoxanide showed significantly decreased viabilities and at the pH in the small intestine. In the same report, the presence of the highest concentration (200 lg/ml) tested, all T. muris died. Addi- active anion form of nitazoxanide was revealed from pH 6 and tionally, nitazoxanide significantly decreased the motility of L3. above (Hoffman et al., 2007). In mice, the mean intestinal pH is On the other hand, differing stage susceptibilities were observed <5.2, much lower than values observed in humans (pH 7.5), with for hookworms; A. ceylanicum L3 were only moderately affected some consistent inter-individual variations (Evans et al., 1988; by nitazoxanide in vitro, while adult worms were killed only at McConnell et al., 2008). If the proposed mode of action for nitazox- the highest tested nitazoxanide dose. The hookworm third stage anide also applies to helminths, the substantial intestinal pH differ- larva is a developmentally arrested non-feeding stage, where phys- ences between humans and rodents might explain the iological processes and exchanges with the environment are unexpectedly low in vivo activity, and would make rodent models greatly reduced (Cassada and Russell, 1975; O’Riordan and Burnell, inappropriate to test drugs strongly relying on narrow intestinal 1989, 1990; Burnell et al., 2005). Hence, if nitazoxanide requires pH ranges. ingestion by the worm to exert its anthelmintic activity, or if the To our knowledge, we have for the first time evaluated the ef- target enzymes are missing at this stage, it would not be surprising fect of nitazoxanide drug combinations. Though synergistic activity that A. ceylanicum L3 would remain unaffected by the drug. Simi- was observed using combinations of nitazoxanide and standard larly, monepantel only affected adult A. ceylanicum and showed a anthelminthic drugs in vitro, these effects could not be confirmed low activity on L3 (Tritten et al., 2011). in vivo, possibly also because of intestinal pH incompatibilities. To our knowledge, we have investigated the effect of nitazoxa- In conclusion, nitazoxanide was found highly potent against nide on A. ceylanicum and T. muris using SEM for the first time. SEM both T. muris stages tested, whereas only A. ceylanicum adult did not identify any marked effect of nitazoxanide (200 lg/ml) on worms were found sensitive to the drug in vitro. In both parasite- the T. muris adult worm tegument compared to controls. However, rodent models, the drug exhibited poor efficacy. Further studies SEM revealed small tegumental knobs in the anterior region of half in non-rodent systems are necessary to explain the differences be- of the treated A. ceylanicum specimen, possibly associated with tween our results and findings obtained in clinical trials. exposure to the drug. Note that, in mice, nitazoxanide is rapidly transformed and only Acknowledgements tizoxanide, the active metabolite, is measurable in plasma. How- ever, in vitro, nitazoxanide and tizoxanide have shown similar J. Keiser acknowledges a personal career development grant activities against a wide range of protozoa and bacteria (Stettler from the SNSF (projects Nos. PPOOA3-114941 and PPOOP3_ et al., 2004; Anderson and Curran, 2007). Surprisingly, however, 135170). We thank Gianni Morson for his great support with the nitazoxanide administered in vivo had no significant impact on SEM work. the helminths infections studied, regardless of the dose and treatment schedule. 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